Uterotropic effects of pregnancy-related estrogens, such as 17beta-estradiol (E2) and estriol (E3), occur by two different mechanisms, involving intracellular and membrane estrogen receptors. Traditionally, steroid hormone activation of intracellular steroid receptors, ligand-dependent transcription factors, leads to changes in transcription occurring over hours to days, described as "genomic actions" of steroid hormones. In contrast, activation of plasma membrane-bound steroid receptors on the cell surface initiates changes in ioR fluxes, second messenger signals, and protein kinase activities in millisecond-to-minute time scale, collectively described as "rapid responses" or "nongenomic actions" of steroid hormones. Whether "rapid responses" ultimately lead to changes in transcription remains an open question. The underlying hypothesis of this proposal is that activation of plasma membrane-bound estrogen receptors by the two pregnancy-related hormones, 17beta-estradiol (E2) and estriol (E3), leads to "rapid responses" which indirectly activate transcriptional regulation and cell proliferation. The research will use new chemically defined reagents constructed with E2 and E3 to isolate cells with membrane-bearing estrogen receptor. Preliminary studies have demonstrated that the reagent with E2 can bind to a subpopulation of MCF-7 cells. The following specific aims will be addressed: (1) Isolation can and characterization of plasma membrane estrogen receptor. Using the new chemically defined reagents of E2 and E3, as "bait," cells with membrane-bearing estrogen receptor will be isolated. Analysis of cell-surface-protein expression pattern of membrane estrogen receptor and non-receptor-bearing cells will identify candidate membrane estrogen-receptors. (2) Examination of biological effects of membrane estrogen receptor signaling. Using the same chemically defined reagents, the effect of membrane estrogen signaling on transcription will be determined, and genes regulated by membrane signaling will be identified using a genome-wide based approach. Finally, the effects of membrane estrogen signally on cell proliferation will be examined. The research will open a new avenue of investigation in which the genome integrates the actions of both membrane and intracellular estrogen receptors.